1. Field of the Invention
The present invention relates to a method of stably preserving an oxygen infusion over a long period of time, as well as a method of manufacturing an oxygen infusion exhibiting a stable preservation over a long period of time.
The oxygen infusion of the present invention is widely applicable in the fields of medicine as well as pharmacy, for example. The present oxygen infusion can, for whole blood transfusion, be used as it is, or with some additives if necessary, in clinical therapies as a substitute for erythrocytes.
2. Description of the Background
Conventional blood transfusion systems which infuse human blood into a blood vessel exhibit various problems including blood type incompatibility, possibility of infection (hepatitis, HIV and the like) and an inadequate shelf-life of erythrocytes which is, i.e., only about 3 weeks. Hence, there has been a great demand for a substitute which can overcome these problems. As one such substitute, an infusion such as an electrolyte infusion and a colloidal infusion are noted, which are widely used at present.
However, these infusions do not exhibit the most essential function of blood, which is, an oxygen-carrying capability, and therefore it is of a great importance to develop an oxygen infusion, i.e., artificial red cells, which can substitute for the oxygen-carrying function of the erythrocyte. Some artificial oxygen infusions have been developed and clinical tests for such oxygen infusions have been advanced. Examples of the oxygen infusions include an aqueous suspension of a perfluorocarbon derivative having high oxygen solubility, a hemoglobin having reversible oxygen bonding ability, such as human hemoglobin, bovine hemoglobin or genetically-engineered hemoglobin; an intra-molecular cross-linked hemoglobin; a water-soluble high-molecular conjugated hemoglobin; and an inter-molecular cross-linked macromolecular hemoglobin. At the same time, however, it has become clear that various types of side effects arise due to the non-cellular structure of these artificial oxygen infusions.
The following are possible reasons why hemoglobin, referred to as Hb hereinafter, is inherently contained in the membranes of erythrocytes.
That is:                1) To suppress the influence of high viscosity and/or colloidal osmotic pressure due to a high-concentration Hb solution having a concentration of 12 to 15 g/dl;        2) To seal Hb having high physiological activity within a membrane, thereby suppressing the escape of hemoglobin;        3) To retain each type of phosphoric acid and glycolysis/reduction enzymes, which are used for maintaining the Hb functions, within the same reaction system; and        4) To obtain an advantage of the cell suspension system, which is non-Newtonian fluid exhibiting a characteristic physiological activity within the blood circulatory system (especially, peripheral blood vessels) due to distinctive fluidity thereof.        
Considering the above-described inherent roles of the erythrocyte structure, it is clear that a suspension system of particles encapsulating hemoglobin therein is preferred as the oxygen infusion.
It is currently known that phospholipids, a component of living organisms, form an vesicle structure by themselves, and Djordjevich and Miller have begun studies of hemoglobin vesicles which utilize liposomes made of phospholipid, cholesterol and fatty acid. Currently, many organizations are conducting studies on the hemoglobin vesicle. The use of a hemoglobin vesicle entails advantages such as: 1) natural hemoglobin can be used as it is; 2) the side effects resulting from hemoglobin can be suppressed; 3) the viscosity, colloidal osmotic pressure and oxygen affinity can be adjusted to arbitrary values, respectively; and 4) the residence (retention) time in circulation system of the living body can be prolonged.
It is known that a heme (protoporphyrin IX), which is an oxygen bonding site of hemoglobin, loses its oxygen bonding capability when it escapes from globin. Thus, it has been well recognized that the stereoscopic frame constructed by globin chains plays a significant role and the hydrophobic field formed therein is important. Consequently, much effort has been dedicated for developing a system which can substitute for the functions of globin.
The present inventors studied various types of porphyrin derivatives and have succeeded in synthesizing a lipid heme (lipid-bonded heme): 5, 10, 15, 20-tetrakis [α, α, α, α-o-{2′,2′-dimethyl-20′(2″-trimethylammonioethyl)phosphonatoxy eicosanamido}phenyl]porphynato-iron (II) and others, which have the capability of bonding with oxygen reversibly in aqueous systems. In a lipid heme vesicle produced by mixing the above lipid heme together with phospholipid, and then dispersing the resulting mixture in an aqueous phase, the lipid hemes are embedded in hydrophobic field of a phospholipid membrane and thus suspended and orientated in the membrane. In a lipid heme vesicle in an aqueous suspension system with a uniform particle size, it has been observed that reversible coordination of oxygen is possible as in the case of hemoglobin in a erythrocyte under physiological conditions. Thus, a red-color aqueous system having the same heme concentration as that of blood appeared as the first oxygen infusion manufactured by total synthesis (E. Hasegawa et al., Biochem. Biophys. Res. Commun. vol. 105, 1416 to 1419, 1982). Bioassay was also carried out extensively by administrating the lipid heme vesicle into animals. In particular, in the resuscitation test for a canine model of hemorrhagic shock, it was confirmed that the lipid heme had oxygen-carrying capability in accordance with the heme concentration. It was further confirmed that a lipid heme-triglyceride microsphere, prepared by covering the outer surface of an microsphere of a nutritional oil material (such as purified soybean oil or triglyceride) with a lipid heme, exhibits an oxygen carrying capability.
Further, another oxygen infusion agent was synthesized which contains 2-[8-{N-(2-methylimidazolyl)}octanolyloxymethyl]-5,10,15,20tetrakis [α, α, α, α-o-pivaloamido]phenylporphynato-iron (II) adsorbed in a hydrophobic pocket of human serum albumin or genetically engineered human albumin, the oxygen infusion agent being referred to as “albumin-heme”, hereinafter. Further, it has been confirmed that the albumin-heme has an oxygen carrying capability (E. Tsuchida et al., Bioconjugate Chemistry, vol. 8, 534-538, 1997).
Thus, considering the current state of such oxygen infusions, one of the principal remaining issues is the preservation thereof.
Methods are known for preserving an oxygen infusion, namely, frozen storage and storage in the form of freeze-dried powder. However, the frozen material requires thawing, which is laborious. On the other hand, the freeze-dried powder requires much time for dissolution in aqueous solution, and further entails the problem of a complicated operation, such as removal of bubbles generated upon dissolution in the solution. Therefore, the frozen storage and freeze-dried powder storage methodologies are not preferred.
In addition, the qualities of oxygen infusions deteriorate with time due to the inherent characteristics of heme protein, and therefore it is difficult to preserve them in a stable condition. More specifically, hemoglobin, lipid heme and heme derivatives can reversibly bond with oxygen when the central iron of heme is a ferrous iron (Fe2+), whereas when the ferrous iron is oxidized to a ferric iron (Fe3+), oxygen binding capability is lost. Further, even a ferrous complex bound with oxygen is gradually oxidized automatically while releasing superoxide anion (O2−), and is finally converted to a ferric iron. Thus, the complex loses its oxygen binding capability (for example, hemoglobin becomes methemoglobin). Further, heme protein thus converted to a met-form can easily release free heme and free ferric iron, which is a concern causing adverse effects on the living body.
Even where preservation is effected in a refrigerator to suppress the above-described oxidation by lowering the reaction rate, the amount of ferric heme gradually increases. In order to solve this problem, a method is known for reducing ferric iron into ferrous iron by adding a methemoglobin-reducing enzyme system which exists in erythrocytes, or an enzyme which can scavenge active oxygen, such as catalase or superoxide dysmutase. Also known is a method of maintaining the ferrous iron by binding carbon monoxide (CO) with heme. The affinity of carbon monoxide to hemoglobin or a heme derivative is as high 200 times that of oxygen, and therefore it is possible to suppress the oxidation to ferric iron for an extremely long period of time.
However, the above-described method in which a methemoglobin-reducing enzyme system or an active oxygen scavenger enzyme is added to the oxygen infusion, entails such drawbacks that the enzymatic activity is lowered during a long period of time and thus the enzymes lose their reduction potential. On the other hand, an oxygen infusion which is preserved in a refrigerator under a carbon monoxide atmosphere can not be directly administered into a human body because a great amount of carbon monoxide contained in the oxygen infusion is extremely harmful, and the oxygen bonding potential of the infusion cannot be exhibited unless the carbon monoxides bound with the heme are removed. For this reason, such a transfusion cannot be given as it is to the human body. In addition, in refrigerator preservation after being converted into an oxy-type, the oxidation to a ferric iron gradually proceeds and eventually the oxygen carrying potential is lowered. The correlation between the oxygen partial pressure of ferrous hemoglobin and the oxidizing rate is well known, and further, it has been experimentally confirmed that the oxidation reaction does not proceed with deoxyhemoglobin (Sakai et al., Bull. Chem. Soc. Jpn., 1994, 1120-1125; Takeoka et al., Bioconjugate Chem., vol. 8, 539-544, 1997).
In addition, even if the oxidation reaction of hemoglobin and heme derivative can be suppressed, the preservation of the oxygen infusion entails another problem. That is, molecular assembly structures, such as a hemoglobin vesicle, a lipid heme vesicle and a lipid heme-triglyceride microsphere which form the environment of heme, are often unstable since these structures are constructed not with covalent bonds but through molecular interaction forces (such as hydrophobic interaction, electrostatic interaction and hydrogen bonds) acting between molecules of the components. As a result, when such an oxygen infusion is suspended in a saline solution and preserved in a refrigerator, the vesicles are fused with each other to form aggregates of the vesicle population, thereby varying the particle diameter thereof. Consequently, there has been a demand for stabilizing the molecular assembly structure of the vesicles. The following is an example of the conventionally known stabilization technique.
Specifically, it is known that a polymerizable phospholipid may be used as a membrane component of a hemoglobin vesicle or a lipid heme vesicle, and the polymerizable phospholipid is polymerized by γ-ray or ultraviolet ray irradiation to highly stabilize the structure of the vesicle. In utilizing this technique, it is possible to preserve the resultant suspension for a long time by rapidly freezing it with liquid nitrogen. Further, even if the freezing and thawing are repeated for 10 times, leakage of hemoglobin, change in the particle diameter or variation in association-dissociation curve of oxygen is not observed (Satoh et al., ASAIO Journal, vol. 38, M580 to M584, 1992). In addition, there can be obtained an extremely stable powder by adding a sugar, such as maltose or sucrose, to the above-described suspension system, followed by freeze-drying the system. For example, for hemoglobin vesicle, it was confirmed that an aqueous suspension of the resultant lyophilized powder showed no leakage of hemoglobin, and no variation in particle diameter thereof, from the physical property analysis carried out on a hemoglobin vesicle which was preserved for 20 weeks at a temperature of 4° C., followed by adding pure water thereto for re-constituting suspension thereof. This indicates that the hemoglobin vesicle is in substantially the same state as that before the lyophilization (Wang et al., Polymer Adv. Technol., vol. 3, 7-21, 1992).
On the other hand, there is a conventionally well known method of introducing a polyoxyethylene-linked lipid onto a surface of a phospholipid vesicle. However, the object of this method is to extend the inblood retention time of the vesicle, thereby efficiently transporting an anticancer agent encapsulated therein to a tumor tissue. This method has already undergone clinical trials and the safety of the method has been fully confirmed. Further, it has been empirically confirmed that the dynamics of the bloodstream can be improved by modifying the surface of a hemoglobin vesicle with polyoxyethylene, which can suppress the interaction between a hemoglobin vesicle and a plasma protein (Sasaki et al., Bioconjugate Chemistry, vol. 8, 23 to 30, 1997). However, it is not known to utilize the polyoxyethylene modification method for the preservation of oxygen infusions, and, as noted above, a need exists for preservation of oxygen infusions.